Traditional vaccination techniques which involve the introduction into an animal system of an antigen which can induce an immune response in the animal, and thereby protect the animal against infection, have been known for many years. Following the observation in the early 1990's that plasmid DNA could directly transfect animal cells in vivo, significant research efforts have been undertaken to develop vaccination techniques based upon the use of DNA plasmids to induce immune responses, by direct introduction into animals of DNA which encodes for antigenic peptides. Such techniques, which are referred to as “DNA immunisation” or “DNA vaccination” have now been used to elicit protective antibody (humoral) and cell-mediated (cellular) immune responses in a wide variety of pre-clinical models for viral, bacterial and parasitic diseases.
Research is also underway in relation the use of DNA vaccination techniques in treatment and protection against cancer, allergies and autoimmune diseases.
DNA vaccines usually consist of a bacterial plasmid vector into which is inserted a strong viral promoter, the gene of interest which encodes for an antigenic peptide and a polyadenylation/transcriptional termination sequence. The gene of interest may encode a full protein or simply an antigenic peptide sequence relating to the pathogen, tumour or other agent which is intended to be protected against. The plasmid can be grown in bacteria, such as for example E. coli and then isolated and prepared in an appropriate medium, depending upon the intended route of administration, before being administered to the host. Following administration the plasmid is taken up by cells of the host where the encoded peptide is produced. The plasmid vector will preferably be made without an origin of replication which is functional in eukaryotic cells, in order to prevent plasmid replication in the mammalian host and integration within chromosomal DNA of the animal concerned.
There are a number of advantages of DNA vaccination relative to traditional vaccination techniques. Firstly, it is predicted that because the proteins which are encoded by the DNA sequence are synthesised in the host, the structure or conformation of the protein will be similar to the native protein associated with the disease state. It is also likely that DNA vaccination will offer protection against different strains of a virus, by generating cytotoxic T lymphocyte responses that recognise epitopes from conserved proteins. Furthermore, because the plasmids are taken up by the host cells where antigenic protein can be produced, a long-lasting immune response will be elicited. The technology also offers the possibility of combining diverse immunogens into a single preparation to facilitate simultaneous immunisation in relation to a number of disease states.
Helpful background information in relation to DNA vaccination is provided in (1), the disclosure of which is included herein in its entirety by way of reference.
Despite the numerous advantages associated with DNA vaccination relative to traditional vaccination therapies, there is nonetheless a desire to develop adjuvant compounds which will serve to increase the immune response induced by the protein which is encoded by the plasmid DNA administered to an animal.
One reason for this is that while DNA vaccines tend to work well in mice models, there is evidence of a somewhat weaker potency in larger species such as non-human primates (2, 3), which is thought to be predictive of the likely potency in humans. Adjuvants may also be useful to correct an inappropriate deviation of immune response from a Th1 to Th2 response which can be associated with DNA vaccination, especially when administered directly to the epidermis (4). Finally, it has been recognised that the DNA itself, through CpG motifs, may exhibit some adjuvant properties (5, 6, 7) which are prevalent in smaller animals administered DNA vaccines intramuscularly, but reduced in larger species or when small amounts of DNA are administered, such as via “gene-gun” administration.
Accordingly, it is one object of the present invention to provide adjuvant compounds which can be used in conjunction with DNA vaccination procedures. It is also an object to provide methods of improved DNA vaccination involving such adjuvants, as well as compositions including the adjuvants concerned. Other objects of the present invention will become apparent from the following detailed description thereof. To date, however, meeting these objects has proven difficult, largely due to mechanistic differences associated with DNA vaccination, as compared to traditional vaccine techniques.
The literature reports numerous instances of humoral immune responses in animal models, which result from DNA vaccination. Antibody responses have been shown against human growth hormone and human α-1 anti-trypsin (8), against influenza NP (9), against HIV Envelope protein (10), bovine herpes virus glycoprotein (11) and hepatitis B surface antigen (12), amongst others, following administration of plasmid DNA encoding therefore. Cytotoxic T-cell responses have also been demonstrated in animal models of DNA vaccination. Generation of cytotoxic T-cells has been demonstrated against NP from influenza A (13), hepatitis B surface antigen (HBs Ag) 65 and core antigen (14), and HIV Env (15, 16, 17) as a few examples. It is interesting to note, however, that helper T-cell response appears to be dependent upon the mode of plasmid DNA delivery. Intramuscular administration biases the helper T-cell response to Th1-like response, whereas administration primarily to the epidermis biases the immune response towards a Th2-like response (4). It is further noted that a number of known immunopotentiating agents have been tried in combination with DNA vaccination techniques with limited, or at best, mixed success. For example, while co-expression of GM-CSF with rabies virus glycoprotein (18) and carcinoembroytic antigen (CEA (19)), and co-expression of B7-1 and B7-2 M. tuberculosis HSP 65 (20) or CEA (19), all induced higher antibody titres than expression of the antigen alone, there is no report of an enhanced cellular immune response. Interestingly also, rabies virus glycoprotein when co-administered with DNA encoding interferon-γ, actually had an inhibitory effect on antibody response (18).
With this background in mind, it is most surprising to note that the present inventors report adjuvant compounds which show the dual action of not only stimulating humoral immune response, but simultaneously stimulating the cellular immune response mechanism. The compounds which have been recently shown to demonstrate this remarkable adjuvant activity in relation to DNA vaccination were disclosed in International Patent Publication No. WO94/07479, in relation to their immunopotentitory activity. One particular compound identified by the present inventors as having favourable DNA vaccine adjuvant activity is 4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid, also known as tucaresol, which was originally described in EP 0054924. None of the compounds now identified by the present inventors as being suited to act as adjuvants with DNA vaccines have previously been disclosed or suggested as demonstrating the humoral and cellular immunogenic activity which so suits them to the role as DNA vaccine adjuvants.